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Customer Solutions

Whether you are shopping around or placing an order, our dedicated Customer Solutions team is available to answer any questions you may have. Submit your inquiry using the form to the right, and a Customer Solutions representative will respond as soon as possible.

Prefer the phone? We're here to help. Call us at 800.592.5726. You can also email cs@alpco.com.

Technical Solutions

Experiencing difficulties or need some assistance while testing one of our products? Contact our Technical Solutions team by submitting an inquiry in the form to the right, and a representative will respond as soon as possible.

Prefer the phone? We're here to help. Call us at 800.592.5726. You can also email ts@alpco.com.

Resources by Application

Browse FAQ's, tools and videos specific to various applications.

Immunoassays

Q. What kind of sample type can be used with the kit I purchased?

A. Each type of kit has been validated for the sample types indicated in its protocol. All other sample types would have to be validated internally. Please contact ALPCO's product support team for further information regarding possible testing of other sample types performed by the product users.

Q. Is this kit cross reactive with another species?

A. Each type of kit has been validated for the species indicated in the protocol. Although cross reactivity may exist, validation of other species would need to be performed internally. Please contact ALPCO's product support team for further information regarding possible species validation performed by the product users.

Q. How many standards and controls do I run?

A. Each kit will include at least one set of standards (or one vial of standard to be diluted) and in most cases one or two controls. Standards and controls (if included) must be run each time the assay is performed and it is highly recommended to run all standards/controls/and samples in duplicate.

Q. Can I use reagents from other kits or lots?

A. No. Do not substitute components from other kits or lots.

Q. Can I modify incubation times or temperatures?

A. No. Please do not deviate from the protocol.

Q. Can I use an expired kit?

A. No. The kit should not be used if it is past the expiration date specified on the kit label.

Q. Can I use a kit that has been left at room temperature?

A. Please be sure that the kit and its components are stored as indicated in the kit insert. For additional information about kit stability at room temperature please contact ALPCO's product support team.

Q. Can I skip running the control(s) on my assay?

A. No. If a control(s) is provided always be sure to run the control(s) with each assay. The concentrations of the controls should fall within the range specified on the certificate of analysis.

Q. Can I wash my plate with a multichannel pipette?

A. We do not recommend the use of a multichannel pipette. Wash buffer must be dispensed with adequate and equal force to properly wash the wells. We recommend the use of a Statmatic wash nozzle www.tricontinent.com/products/statmatic-i/ or an automated plate washer. Please refer to the Learning Center tab of the Resources section of the website for a video displaying proper washing technique or click here.

Q. Is it necessary to make a layout of my samples?

A. It is extremely important to know where your standards, controls and samples are located on the plate so that results are generated appropriately. Click here for a downloadable platemap. It also helps to label the strips on the plate (use small tabs at each end) in the event that strips become loose while decanting.

Q. Why is the washing step so important?

A. Correct washing of the plate is a very critical and important aspect of running any successful ELISA. Consistency in washing the plate is essential. Washing the plate too rapidly or too slowly, incomplete washing or aspirating, and allowing the wells to sit dry are all factors that will affect the precision of the assay. We do not recommend the use of a multichannel pipette for this purpose. Multichannel pipettes do not apply enough pressure to thoroughly remove the unbound material and this will result in higher background noise. A video displaying proper washing technique is available in the Learning Center section of the website or click here.

Q. How many times can I freeze and thaw my samples?

A. Recommended freeze/thaw cycles vary based on kit. We recommend to avoid repeated freeze and thaw cycles unless otherwise indicated in the manual. If the collected sample amount allows for it, prepare and freeze aliquots to eliminate or reduce freeze/thaw cycles.

Q. Can I use my own "homemade" buffers?

A. Each kit contains diluents and buffers that are formulated to closely match specific sample types. The manufacturer will not guarantee the performance of the kit if components that were not provided with the kit are used to run the assay.

Q. What should I do if I run out of diluent?

A. Enough reagents are provided to run the entire kit when the protocol is followed. If your type of study requires a greater dilution, calculate the amount of diluent needed prior to setting up the assay and additional diluent may be available for purchase. Please contact customer support for information on the availability of individual components.

Q. My controls are out of range. Is my data invalid?

A. The concentration of the controls must be within the range specified in the certificate of analysis. If the controls are out of range the assay may be invalid. Also, make sure the curve fit recommended in the protocol has been used. If more than one recommendation is provided use the regression method that best fits the standard data points.

Q. I didn't get good values from my standard curve. Can I use the example values from the protocol?

A. No. Never use example values to calculate your assay results. Sample concentrations should be generated from the standard/calibrator values obtained with each assay.

Q. My samples generated OD values out of the standard range. Can I still calculate concentrations?

A. Only sample values that fall within the range of the standard curve can be used. Values outside of this range are generally not linear and can lead to incorrectly extrapolated results.

Q. My standard curve is fine but I didn’t get a signal with my samples. Why?

A. The sample may contain the analyte but it is undetectable based on sensitivity of the kit. The matrix of the sample may also be masking the detection. Ensure that the dilutions have been made as stated in protocol and review the procedure to ensure all reagents were added with the correct volume and in the correct order.

Q. How many samples can I run?

A. ALPCO now offers a tool to help with calculating the number of samples that can be run in a kit. Once you review the protocol to determine the number of standards, controls, and blanks that may be required for the specific kit you plan to run click here to go to the Sample Calculator.

Q. What is reference wavelength?

A. A reference wavelength is a secondary wavelength used for correction (normalization) of the principal wavelength OD values. It helps to account for imperfections in the plate.

Q. Why is 450nm the most common measure wavelength?

A. The measurement wavelength is determined by the substrate used in each kit. The most common one is TMB. It produces a blue color measurable at a wavelength of 650nm. It can be used in end point assays by stopping the reaction with either 1M phosphoric acid or 1M sulfuric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm.

Q. What is the difference between measurement wavelength and reference wavelength?

A. The main difference between measure wavelength and reference is that the first one has been chosen to read the absorbance produced by the substrate and the second one (reference) to detect imperfections in the plate.

Q. Can I read the plate without having a reference wavelength?

A. Yes, you can. However, the use of a reference wavelength may improve the sensitivity of the assay.

Poor precision between duplicates (high CV values)

Automated plate washer is not functioning properly and is not washing all wells in the same manner:

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available here click here .

Problem with the multi-channel pipette during the addition of the conjugate or substrate:

Check pipette calibration and for bubbles during pipetting. Practice pipetting technique. A video providing tips on proper pipetting technique is available here .

Poor standard curve, does not match certificate of analysis

Inadequate plate washing:

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here.

Poor pipetting technique:

Check pipette calibration and for bubbles during pipetting. Practice pipetting technique. A video providing tips on proper pipetting technique is also available in the Learning Center section of the website or click here.

Inadequate mixing of reagents:

Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc) to ensure reagents are homogenous before use. Confirm the correct volumes were used if mixing kit reagents to make a “working” component for the assay.

Inadequate shaking of plate:

Incorrect shaking motion can affect proper mixing and antibody/analyte interaction. Due to various equipment models please refer to the Learning Center section of the website for a diagram indicating the appropriate shaking method or click-here.

Wrong volume of reagents added to the well:

Check residual volumes to confirm correct volume of reagents were added to the wells.

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here

Contamination of the substrate with enzyme conjugate:

Ensure all containers are clean and clearly marked. Check TMB for blue coloring. If TMB has turned blue, do not use in your assay and contact product support. Also do not expose substrate to light before use.

High background OD readings (Blank or 0 STD values are too high)

Automated plate washer is not functioning properly and is not washing all wells in the same manner:

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here

Omission of a wash step

Old or contaminated wash buffer:

Make fresh wash buffer.

Contamination of the substrate with enzyme conjugate:

Ensure all containers are clean and clearly marked. Check TMB for blue coloring. If TMB has turned blue, do not use in your assay and contact product support. Also do not expose substrate to light before use.

Wrong conjugate dilution:

A higher than normal concentration may result in an elevated background. Check residual volumes and confirm conjugate was prepared correctly.

Wrong filter used in the plate reader:

Confirm the correct filter indicated in the protocol is being used when reading the plate.

Follow method in protocol for appropriate sample prep or call ALPCO tech support for clarification.

Omission of a reagent:

Check residual volumes to confirm correct volume of each reagent was added to the wells.

Dilution error:

Double check dilution calculations and the volumes used in making these dilutions. If needed, remake the dilutions.

Sample IDs mixed up:

Review sample ID’s to confirm they were labeled correctly.

Wrong curve fit used:

Use the curve fit recommended in the protocol. If more than one recommendation is provided use the regression method that best fits the standard data points. Also, it is very difficult to generate a 4 or 5 parameter curve with Excel and the use of a software program for this type of regression is recommended.

No color development or very low OD readings

Wrong filter used in the plate reader:

Confirm the correct filter indicated in the protocol is being used when reading the plate.

Inadequate mixing of reagents:

Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc) to ensure reagents are homogenous before use. Confirm the correct volumes were used if mixing kit reagents to make a “working” component for the assay.

Inadequate shaking of plate:

Incorrect shaking motion can affect proper mixing and antibody/analyte interaction. Due to various equipment models please refer to the Learning Center section of the website for a diagram indicating the appropriate shaking method or click-here.

Reagents contaminated or added in the wrong order:

Ensure all containers are clean and clearly marked and follow the steps as instructed in the protocol.

Assay incubation times not followed correctly:

Follow the incubation times and temperatures as indicated in the protocol. Modifying either factor will affect the assay kinetics and may lead to erroneous results.

Improperly prepared sample:

Follow method in protocol for appropriate sample prep or call ALPCO tech support for clarification.

Verify the appropriate standard curve was used and check results against the values given in the certificate of analysis.

Incorrect reconstitution volume used:

Follow protocol as instructed. Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc.) to ensure reagents are homogenous before use.

Wrong curve fit used:

Use the curve fit recommended in the protocol. If more than one recommendation is provided use the regression method that best fits the standard data points. Also, it is very difficult to generate a 4 or 5 parameter curve with Excel and the use of a software program for this type of regression is recommended.

A. Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.

Q. How are fluorophores conjugated to antibodies?

A. There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.

Q. What controls should I use for a flow cytometry experiment?

A. Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the auto fluorescence associated with any particular cell type.

Q. How do I know which fluorophores are compatible with my instrument?

A. Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.

Q. What is compensation?

A. Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal "bleeds" into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.

Q. How does fixation affect fluorophores?

A. Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.

Q. What is a tandem dye?

A. Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET).

Q. Should I use the amount of antibody recommended by the protocol?

A. I would always recommend performing a titration because sometimes you find you can use less antibody than what is recommended for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands. The % positive staining should be maintained with concentration selected - i.e. if while they are titrating they find the population % drops then they are not staining all the cells and should use a higher concentration.

Reducing non-specific binding

Non-specific binding can be due to several reasons.

a) Too much antibody can increase the amount of non-specific binding of your negative population reducing your signal:noise. If the antibody you are using has not been titered, then a titration of your antibody should be done to determine the optimal concentration.

b) Non-specific binding can also be due to Fc-mediated binding. You can use IgG (Lampire) of the same species as your antibody to block non-specific binding. Using monoclonal antibodies specific for Fc Receptors to block Fc-mediated binding can also help reduce background binding.

c) The use of directly conjugated antibodies can also reduce the amount of non-specific binding.

d) The use of a biotinylated antibody with a streptavidin fluorochrome conjugate can be a source of non-specific binding in some cells. Biotin is a component of normal cellular metabolism, and as such, this may be detected in high levels in cells.

e) Dead cells are notorious for non-specifically binding antibodies. Inclusion of a viability dye (i.e. PI, 7-AAD) in your assay will allow you to exclude the dead cell population from your analysis. You must make sure that your viability dye is compatible with the other fluorochromes in your sample. Also, cells cannot be fixed when using a viability dye as this will make them permeable to the dye and all the cells will appear dead.

Infinicyt™: Flow Cytometry Software

Infinicyt™ is a flow cytometry analysis software program that provides an innovative approach for data integration and multidimensional analysis of flow cytometry data. Its state-of-the-art features make analysis and interpretation of results easier, faster and more accurate.

Infinicyt™ is a flow cytometry analysis software program that provides an innovative approach for data integration and multidimensional analysis of flow cytometry data. Its state-of-the-art features make analysis and interpretation of results easier, faster and more accurate.

The new Infinicyt™ 1.7 version features:

Maturation (New tool)

Batch Analysis

Compass

Multiple ways of data display

APS (Automatic Population Separator)

Analysis Strategy

Reference Image

File Merge

Calculate Data

Customizable Report

HPLC/LC-MS

Q. What are the basic components of an HPLC system?

A. A functioning HPLC system includes a sampler, pump, column, detector and data processor. A degasser and column oven may also be used.

A. Check the assay protocol to instructions on the appropriate column. The most commonly used column is C18 which covers a wide range of applications.

Q. How do I make a column last longer?

A. Filter samples to remove particulate and make sure the pH of the mobile phase is within specifications for the column. If the column will be stored for an extended period of time, flush with methanol or acetonitrile.

There may be contamination at the head of the column. Change direction of the column and rinse for 30 minutes at a low flow rate (0.2ml/min) with mobile phase.

There may be air in the system. Degas pump.

Vials may be contaminated. Use new vials or clean with methanol.

Variable retention times

There may be a drift in temperature. Use a column oven.

Imprecise pump delivery. Check pumps and degas system if needed.

System is not in steady state yet. Rinse system mobile phase for 15 minutes.

Baseline is drifting

Detector lamp may not have reached working temperature. Wait until appropriate temperature is reached.

Detector lamp may be too old and needs to be replaced.

System is not in steady state yet. Rinse system mobile phase for 15 minutes.

Imprecise pump delivery. Check pumps and degas system if needed.

Baseline is not smooth

Imprecise pump delivery. Check pumps and degas system if needed.

Detector flow cell is dirty. Try cleaning flow cell.

Chemiluminescence

Chemiluminescent Plate Reader Settings

Reader Settings

Please contact the microplate reader manufacturer’s technical services department for additional assistance. The instrument settings below are meant to serve as a guideline. It is optional to shake the plates before reading for less than or equal to 3 seconds.